Oxygen is essential for life, and an enormous amount of medical treatment centers around normalizing or stabilizing oxygen levels in the body. Evidenced by the explosion of new pulse oximetry and near infrared spectroscopy (NIRS) devices on the market, optical spectroscopy is attractive for noninvasive tissue monitoring in many hospital settings. But we are not there yet - no existing clinical device has overcome the critical hurdle of accurately quantifying cellular biomarkers that are sensitive to oxygenation. We propose to develop a novel, noninvasive cell oximeter for use in humans that will provide snapshots of tissue oxygen profiles (TOPs) in muscle by simultaneously measuring hemoglobin saturation, myoglobin saturation, and cytochrome aa3 oxidation from optical reflectance spectra. Three different compartments will be interrogated with light: capillaries, myocytes, and mitochondria. In this proposal we focus on skeletal muscle, but the general approach is applicable to any tissue that is close enough to the probe surface to be interrogated with light. TOP monitoring will open windows for assessment of oxygen availability, delivery, and consumption. Clinicians will be able to identify cellular oxygen insufficiency and will be able to target treatment better when they can localize oxygen deficits along the metabolic pathway. The specific aims of this proposal are to: 1) develop spectral preprocessing steps to remove skin and lipid absorbances and scattering effects; 2) develop and validate measurement of TOP by Locally Weighted Regression (LWR) in vitro; and 3) develop and validate measurement of TOP by LWR in vivo. Aims 1 and 2 will be accomplished in vitro using phantoms. A new probe will be built to measure spectra from superficial and deep tissue regions. Spectral preprocessing steps will be developed and tested with phantoms that have known hemoglobin concentrations. The goal of spectral preprocessing is to remove absorbances due to skin and fat and scattering effects from spectra, leaving spectra that primarily contain absorbance information relevant to TOPs. TOP measurement by LWR will be developed in vitro with phantoms that model a human hand. Finally, in Aim 3, spectra will be acquired from the hands of healthy subjects during arm ischemia. These spectra will be used to calibrate in vivo LWR models. The models will enable TOP monitoring in real time from new spectra collected from measurement sites that are accessible from the skin surface (e.g., hand, arm, leg, and brain). Tracking oxygen from blood to mitochondria with our new device will have broad implications in clinical practice and clinical research. We envision that TOP monitoring will become a critical component in the care of patients with oxygen insufficiency, including those with sepsis, shock, and cardiac failure. Our long-term goal is to develop the cell oximeter for clinical use in ambulances, emergency departments, intensive care units, and operating rooms.